The development of a protective vaccine remains a high priority for the global control of the HIV/AIDS epidemic. However, the unique biological features of HIV-1 make this task extremely challenging. The main obstacles include the ability of the virus to integrate, a remarkable degree of genetic variability, and the cryptic, antibody-shielded conformation adopted by the viral envelope. The inherent conformational flexibility of gp120, along with the extensive N-linked glycosylation that covers most of the exposed surface of the glycoprotein, has hampered attempts to elucidate the native structure of the HIV-1 envelope spike. Thus, most high-definition structures of gp120 were obtained with deglycosylated, variable loop-truncated core monomers in complex with stabilizing ligands such as CD4. Although new insights have recently come from the crystal structure of a stabilized soluble cleaved gp140 SOSIP trimer, many critical aspects related to the mechanisms of HIV-1 immune vulnerability and evasion remain unresolved. In particular, the fine molecular details of the interaction between the V2 and V3 loops of gp120, which play a critical role in stabilizing the pre-fusion envelope structure, are only partially elucidated. We recently made a series of observations that shed new light on the structure of the variable loops in the native HIV-1 envelope. 1. The conserved central region of V2 contains sulfated tyrosines. We recognized that the conserved central region of V2 contains two tyrosine residues (Tyr173 and Tyr177) with identical spacing as two tyrosines present in the N-terminal region of CCR5 (Tyr10 and Tyr14), which were shown to be post-translationally modified by O-sulfation and to play a critical role in the interaction of CCR5 with V3. Thus, we investigated whether the V2 tyrosines can also be sulfated. Analysis by Western blot using a highly specific anti-sulfotyrosine (Tys) mAb documented the presence of sulfated tyrosines in gp120 immunoprecipitated from the surface of HeLa cells expressing full-length cleavable gp160 from the subtype-B HIV-1 isolate BaL. Since gp120 contains additional tyrosine residues both within and outside V2, we produced a partial V2-deletion mutant that selectively excludes Tyr173 and Tyr177, showing that the Tys signal was abrogated in the deletion mutant. The presence of sulfated tyrosines in gp120 and their selective localization in the V2 loop were also confirmed by metabolic labeling with free 35Ssulfate, as well as by mutagenesis of Tyr173 and Tyr177 both individually and in combination. 2. Continuous cell lines show low constitutive levels of tyrosine sulfation. Continuous cell lines are generally inefficient in post-translationally modifying tyrosines by O-sulfation. We confirmed this finding by showing that the constitutive levels of tyrosine sulfation in CHO-expressed recombinant gp120 (BaL) were low, although they could be markedly enhanced by overexpression of the sulfotransferase TPST2, while treatment with the TPST inhibitor NaClO3 (30mM) totally abrogated the signal. These findings may have important implications because continuous cell lines are commonly utilized to produce gp120 in various settings, including vaccine clinical trials and neutralization assays. 3. Virion-associated gp120 produced by primary CD4+ T cells is efficiently sulfated. Next, we tested the levels of V2 tyrosine sulfation in gp120 purified from HIV-1 virions produced by primary human CD4+ T lymphocytes, which are physiologically relevant target cells for HIV-1 infection. When identical amounts of serially diluted virion-associated gp120 from HIV-1 BaL and a reference tyrosine sulfated antibody (mAb 412d) were loaded onto the same gel, both proteins displayed high levels of tyrosine sulfation. These results were confirmed on 6 unrelated HIV-1 isolates produced in primary CD4+ T cells, including both laboratory-expanded and primary isolates displaying different coreceptor-usage phenotypes: 3 were CCR5-tropic (BaL, JR-FL, ADA), 2 dual-tropic (92US077, 92HT599) and 1 CXCR4-tropic (IIIB). Altogether, these data demonstrate that in different HIV-1 isolates grown in physiologically relevant target cells the V2 tyrosines are efficiently sulfated, in sharp contrast with the inefficiency documented in continuous cell lines. 4. A tyrosine-sulfated V2 peptide mimetic interacts with the CCR5-binding site at the base of V3. Having established that the V2 loop of gp120 contains sulfated tyrosines with identical spacing as in the CCR5 N-terminus, we hypothesized that these modified tyrosines could functionally mimic the CCR5 sulfotyrosines and mediate intramolecular interaction of V2 with the base of V3 in the CD4-unbound pre-fusion gp120 state. This interaction is compatible with the recently published crystal structure of a soluble SOSIP gp140 trimer, where the 173-177 segment is seen directly juxtaposed to the CCR5-binding region at the base of V3, even though sulfotyrosines were not detected in this structure, most likely because the trimer was produced in HEK293 cells. Since the sulfated human mAb 412d interacts with the CCR5-binding site in V3 in a similar fashion as the CCR5 N-terminus, we tested the ability of a tyrosine-sulfated 18aa. peptide mimetic derived from the central region of V2 (pV2&#945;-Tys; aa. 168-185, bearing sulfations on both Tyr173 and Tyr177) to compete with 412d binding to gp120-BaL by surface plasmon resonance. The peptide potently inhibited binding of surface-bound mAb 412d to CD4-activated gp120, while its unsulfated counterpart (pV2&#945;) had a limited effect. The interaction of the V2 peptide mimetic with the 412d-binding site was confirmed using a virion-capture assay in which the HIV-1 envelope is displayed on the surface of intact virions. 5. Tyrosine sulfation in V2 specifically modulates gp120 epitope exposure and neutralization sensitivity. To investigate the functional role of the V2 sulfotyrosines, we tested the effects of modulation of tyrosine sulfation on gp120 epitope accessibility and neutralization sensitivity using a panel of antibodies to the major neutralization regions of gp120 and monomeric sCD4. A striking dichotomous effect was observed on the antigenic profile of gp120 from both the BaL and YU2 isolates. Enhancement of tyrosine sulfation by TPST2 overexpression dramatically reduced binding of monomeric sCD4 (receptor-binding site), mAb 412d (coreceptor-binding site) and anti-V3 loop mAbs B4e8 and D19, all ligands with a restricted binding capacity to the native trimeric spike; in contrast, TPST2 overexpression markedly increased recognition by the trimer-preferring antibodies PG9, PG16, CH01 and PGT145, which are directed to quaternary, glycan-dependent V2 epitopes that are stabilized on the native trimer. Opposite effects were observed when tyrosine sulfation was inhibited by NaClO3 treatment, with a marked increase of sCD4, 412d and anti-V3 antibody binding and a reciprocal decrease in recognition by trimer-preferring antibodies. Strikingly, the pattern of epitope exposure observed by flow cytometry was precisely mirrored by the results of neutralization studies. TPST2 overexpression increased resistance of HIV-1 BaL to neutralization by sCD4, 412d and anti-V3 antibodies, but at the same time it made the virus more sensitive to neutralization by PG9, PG16, CH01 and PDT145; on the contrary, inhibition of V2 tyrosine sulfation by NaClO3 treatment increased neutralization by sCD4, 412d and anti-V3 antibodies, while decreasing sensitivity to trimer-preferring antibodies. These results suggest that the sulfotyrosine-mediated V2-V3 interaction plays a critical role in stabilizing the trimeric envelope conformation and specifically modulates the sensitivity of HIV-1 to antibody-mediated neutralization.